Enantioselective Protein-Sterol Interactions Mediate Regulation of Both Prokaryotic and Eukaryotic Inward Rectifier K+ Channels by Cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K+ (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by 86Rb+ influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket

N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (KATP) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (Po) and bursting patterns of activity observed in full-length KATP channels. However, the nature of TMD0–Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-KATP channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP2. Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP2. Moreover, they uncover a novel mechanism of KATP channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2

A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications

AIMS/HYPOTHESIS: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects. METHODS: The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes. RESULTS: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients. CONCLUSIONS/INTERPRETATION: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response

PIP2-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations†

Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains

Molecular and Electrophysiological Characterization of a Novel Cation Channel of Trypanosoma cruzi

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of a novel cation channel (TcCat) from Trypanosoma cruzi, the etiologic agent of Chagas disease. This channel is potassium permeable and shows inward rectification in the presence of magnesium. Western blot analyses with specific antibodies indicated that the protein is expressed in the three main life cycle stages of the parasite. Surprisingly, the parasites have the unprecedented ability to rapidly change the localization of the channel when they are exposed to different environmental stresses. TcCat rapidly translocates to the tip of the flagellum when trypomastigotes are submitted to acidic pH, to the plasma membrane when epimastigotes are submitted to hyperosmotic stress, and to the cell surface when amastigotes are released to the extracellular medium. Pharmacological block of TcCat activity also resulted in alterations in the trypomastigotes ability to respond to hyperosmotic stress. We also demonstrate the feasibility of purifying and reconstituting a functional ion channel from T. cruzi after recombinant expression in bacteria. The peculiar characteristics of TcCat could be important for the development of specific inhibitors with therapeutic potential against trypanosomes

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

An unusual mechanism of ion channel regulation generates voltage-dependent gating in the absence of a canonical voltage-sensing domain

Differential lipid dependence of the function of bacterial sodium channels

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative 22Na+ uptake into proteoliposomes containing a 3:1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different “guest” glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains

Genetički polimorfizmi u dijabetesu: Utjecaj na terapiju oralnim antidijabeticima

Due to new genetic insights, etiologic classification of diabetes is under constant scrutiny. Hundreds, or even thousands, of genes are linked with type 2 diabetes. Three common variants (Lys23 of KCNJ11, Pro12 of PPARG, and the T allele at rs7903146 of TCF7L2) have been shown to be predisposed to type 2 diabetes mellitus across many large studies. Individually, each of these polymorphisms is only moderately predisposed to type 2 diabetes. On the other hand, monogenic forms of diabetes such as MODY and neonatal diabetes are characterized by unique clinical features and the possibility of applying a tailored treatment. Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to interindividual differences in the efficacy and toxicity of a number of medications. Mutations in genes important in drug absorption, distribution, metabolism and excretion (ADME) play a critical role in pharmacogenetics of diabetes. There are currently five major classes of oral pharmacological agents available to treat type 2 diabetes: sulfonylureas, meglitinides, metformin (a biguanide), thiazolidinediones, and α-glucosidase inhibitors. Other classes are also mentioned in literature. In this work, different types of genetic mutations (mutations of the gene for glucokinase, HNF 1, HNF1ß and Kir6.2 and SUR1 subunit of KATP channel, PPAR-γ, OCT1 and OCT2, cytochromes, direct drug-receptor (KCNJ11), as well as the factors that influence the development of the disease (TCF7L2) and variants of genes that lead to hepatosteatosis caused by thiazolidinediones) and their influence on the response to therapy with oral antidiabetics will be reviewed.Dijabetes tipa 2 dosegao je proporcije epidemije u SAD (> 18 milijuna) i cijelom svijetu (170 milijuna oboljelih osoba) te ima tendenciju daljnjeg dramatičnog rasta. Stoga se u posljednje vrijeme ulažu napori da se otkriju i razviju novi farmakološki agensi za liječenje ove bolesti. Klasifikacija šećerne bolesti proširena je uspjesima istraživača na području genetike. Da bismo razumjeli farmakogenetiku antidijabetika neophodno je razumjeti genetiku samog dijabetesa. Kao što će biti prikazano u ovom radu veliki broj gena koji su povezani s razvojem dijabetesa takođe utječu i na odgovor na terapiju antidijabeticima. S druge strane, mutacije gena koji utječu na ADME (apsorpcija, distribucija, metabolizam i ekskrecija) lijeka imaju značajan utjecaj na farmakogenetiku oralnih antidijabetika. Utvrđeno je da je dijabetes genetički heterogena bolest. Uobičajeni oblici dijabetesa su gotovo uvijek poligenski i za razvoj same bolesti vrlo su značajne snažne interakcije među različitim genima kao i između gena i okoliša. Zbog toga mutacije ili polimorfizmi koji u manjoj mjeri utječu na funkciju gena mogu postati klinički značajni samo u slučaju kada se kombiniraju s drugim faktorima odnosno genima. Smatra se da u razvoju dijabetesa mogu sudjelovati stotine pa čak i tisuće gena. Do 2006. identificirano je nekoliko uobičajenih alela koji povećavaju rizik za razvoj dijabetesa, od kojih su najznačajniji PPARG (Pro12), KCNJ11 (Lys23) i TCF7L2 (T na rs7903146). Do danas je najveći uspjeh postignut u identifikaciji gena odgovornih za razmjerno rijetke oblike ove bolesti poput ”Maturity-onset diabetes of the young” (MODY) i neonatalnog dijabetesa. Monogenske oblike dijabetesa odlikuju jedinstvene kliničke karakteristike i mogućnost primjene individualnog tretmana. Genetički polimorfizmi enzima koji utječu na metabolizam lijekova, transportera, receptora i drugih ciljeva djelovanja lijekova povezani su s interindividualnim razlikama u efikasnosti i toksičnosti mnogih lijekova. Vrlo je važno da se na temelju farmakogenetičkih istraživanja mogu predvidjeti neki neželjeni efekti lijekova. Trenutačno postoji pet glavnih klasa oralnih antidijabetika: sulfoniluree, meglitinidi, metformin (bigvanid), tiazolidindioni i inhibitori α-glukozidaze. U literaturi se također spominju inhibitori dipeptidil peptidaze IV (DPP-IV), selektivni antagonisti kanabinoidnog receptora 1 (CB-1), glukagonu slični peptid 1 mimetici i amilin mimetici. Razumijevanje mehanizama koji rezultiraju disfunkcijom β stanica na fiziološkom i molekularnom nivou neophodno je za napredak u razumijevanju tretmana dijabetesa. U ovom radu dat je pregled različitih genetičkih mutacija (mutacije gena za glukokinazu, HNF 1, HNF1ß, Kir6.2 i SUR 1 podjedinicu KATP kanala ß stanica, PPAR-γ, OCT1 i OCT2, citohrome, KCNJ11, faktore koji utječu na razvoj bolesti (TCF7L2) i varijante gena koji dovode do hepatosteatoze uzrokovane tiazolidindionima) te njihov utjecaj na odgovor na terapiju oralnim antidijabeticima